The main goal of the proposed research is to understand the mechanism of chromosomal DNA replication in eukaryotic cells. The emphasis of the project is on the reconstitution of the yeast DNA replication fork and an intensive study of several of its components by genetic and biochemical means. With the knowledge that several replication proteins are also required for a normal damage response in the cell, another area of interest is to understand this response at the DNA level and to study the subsequent remodeling of the replication fork. The four specific aims in the proposal are: (1) X-ray crystallographic studies of Replication Factor C (RF-C), a 5-subunit complex which loads the replication clamp PCNA, and of sub-complexes of RF-C and alternative RF-C-like complexes which are involved in damage response in the cell; (2) Mechanistic studies of RF-C to understand how this complex loads PCNA and functions during Okazaki fragment synthesis; (3) Structure-function studies of PCNA in DNA replication and DNA repair; (4) Reconstitution and study of RF-C-like complexes which may have specific functions during Okazaki fragment synthesis, chromosome segregation, and damage response in the cell. Because the components of the replication machinery and the damage response machinery are conserved in eukaryotes the principal investigator expects that an understanding of the mechanism and regulation of these machineries in yeast will serve as a model for understanding these processes in human cells. Improper function and regulation of these processes in humans may lead to the accumulation of mutations and cancer.